The eradication of global pathogens, including malaria, hinges upon the development of effective vaccines. One of the key strategies to realize successful vaccines is to identify and develop a new mechanism-based adjuvant. ?-galactocylceramide (?-GalCer), a glycolipid that has been extensively investigated, is known to display a significant biological activity, including an adjuvant effect, by binding CD1d molecules and stimulating invariant NKT (iNKT) cells, an important sub-population of the immune response. Recently, we identified a novel ?-GalCer analog, 7DW8-5, which can strongly enhance the level of malaria-specific protective immune response upon its intra-muscular (i.m.) co-administration with a candidate malaria vaccine. When compared to ?-GalCer, its parental glycolipid, we found the adjuvant effect of 7DW8-5 to be clearly superior. 7DW8-5 exhibited not only a stronger stimulatory activity towards iNKT cells and dendritic cells (DCs), but also a higher binding affinity to both mouse and human CD1d molecules than ?-GalCer. Very surprisingly, we discovered that when administered by i.m. injection, only ?-GalCer, but not 7DW8-5, induced a systemic production of cytokines including IFN-? and IL-12, whereas both glycolipids induced a similar level of systemic cytokine production upon their intravenous administration. We hypothesize that 7DW8-5 is retained locally at the i.m. injection site and nearby draining lymph nodes due its higher binding affinity to CD1d molecules and/or the up- regulation of CD1d expression level on activated DCs by 7DW8-5. In Aim 1, therefore, we will first determine whether 7DW8-5 is indeed retained locally and if so, how 7DW8-5 is prevented from spreading out and inducing systemic cytokine production. We also suspect that its local retention is directly related to the superior adjuvant effect exerted by 7DW8-5 and hypothesize that an increased activation of DCs induced by 7DW8-5 results in a stronger onset of the adaptive immune response elicited by vaccines. We will test this hypothesis in Aim 2. Finally, it is still unclear whether 7DW8-5 could indeed enhance humoral and CD4+ T cell responses and whether these responses could contribute to the protection against malaria. Therefore, using mice lacking CD8+ T cells and/or antibodies, we will determine these issues in Aim 3. In view of our recent studies showing that 7DW8-5 provides a significant adjuvant effect on the cellular immunogenicity of an adenovirus-based malaria vaccine in non-human primates, and because 7DW8-5 is advancing into a Phase 1 clinical trial in 2012, the proposed studies should be imperative for the future clinical applications of 7DW8-5 not only as an adjuvant for various vaccines, but also as a direct therapeutic agent. PUBLIC HEALTH RELEVANCE: Malaria continues to pose a grave threat to the global community and in particular to adults and children traveling to or living in tropical and subtropicl regions of the world. The purpose of this proposal is to study and understand how the efficacy of malaria vaccines gets enhanced by employing glycolipids as immuno- enhancing compounds or adjuvants. The development of a suitable malaria vaccine/glycolipid adjuvant combination would have the potential to decrease the incidence of malaria and diminish the morbidity and mortality attributable to this pathogen.